This proposal is designed to help understand the role played by the pteridine cofactor, tetrahydrobiopterin, in the mixed function oxygenase reactions of phenylalanine and tyrosine hydroxylase. During the course of these reactions one atom of molecular oxygen is inserted into the aromatic ring of phenylalanine or tyrosine with a synchronous conversion of the tetrahydropterin to an "active" or "dihydro quinonoid" form of the pterin. Pterin reductase, in the presence of NADH, converts the dihydropterin back to the tetrahydro level. With the aid of affinity chromatography, it should be possible to isolate the three enzymes in homogeneous form from sheep liver and brain. Their main structural features will then be examined with the use of polyacrylamide electrophoresis, gel filtration, specific amino acid blocking and circular dichroism techniques. Analogs of the amino acid substrates containing photoaffinity labels will be synthesized to aid in the characterization of the hydroxylase active sites, and in addition, previously synthesized fluorescent folate analogs will be used to probe the pterin binding site of the reductase. Improved assay procedures for the enzymatic reactions using fluorescent and fast-reaction techniques are described, together with chemical and kinetic experiments designed to determine whether the tetrahydropterin cofactor participates in the coupled enzymatic reaction as an oxygen/hydroxyl carrier or merely as a reducing agent for the hydroxylases. As a corollary to this program, alternate sources for the hydroxylases are considered, and a new technique for the separation of 6- and 7-substituted tetrahydropterins is proposed.